S-1 nuclease mapping (Hamada et al., BBRC 170, 53, 1990) and cDNA sequencing (Babij et al., PNAS 88, 10676, 1991) revealed that intestinal myosin contains an insert of 7 amino acids (aa) in the head region (25 kD-50 kD junction) of the myosin heavy chain (MHC) near the ATP binding site. A different insert of 10 aa is present at this exact location in certain brain MHCs (Takahashi et al., JBC, in press 1992). Using RT- PCR, we compared avian gizzard and aortic myosins with respect to the presence of this 7 aa insert. We found that both the 204 kD and 200 kD MHCs of gizzard contain this insert, but neither of the aortic MHCs. Moreover, we found no evidence for other inserts in either MHC and SDS- PAGE revealed no differences in the relative content of the 204 kD and 200 kD MHCs between gizzard and aorta. Evidence that the 7 aa insert alters the activity of gizzard myosin is: a two-fold increase in both the Vmax of the actin-activated Mg2+ATPase activity and the velocity of movement of actin filaments in an in vitro motility assay, compared to aorta. SDS-PAGE and urea-PAGE revealed two isoforms of the 17 kDa light chain (LC17) in aorta, but only one in gizzard myosin. Exchange of gizzard LC17 onto aorta MHC resulted in no change in the in vitro motility assay suggesting that the differences in enzyme activity are due to the 7 aa insert.